Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Rational Design of Hemoproteins for Peroxidation Reactions(01. Izmir Institute of Technology, 2020) Güralp, Gülce; Güralp, Gülce; Sürmeli Eraltuğ, Nur Başak; 01. Izmir Institute of TechnologyBiocatalysts are important for the synthesis of fine chemicals and steroidal drugs in the biopharmaceutical industry. Cytochromes P450 (P450) monooxygenases are significant biocatalysts due to their high selectivity for oxidation reactions. CYP119 is the first characterized thermoacidophilic P450. CYP119 was isolated from Sulfolobus acidocaldirius. CYP119 enzyme shows high stability at low pH and high temperature. CYP119 can utilize the peroxidase shunt pathway in the catalytic cycle of P450. These abilities make CYP119 attractive biocatalyst for production of fine chemicals and drugs. In this study, Leu69Gly mutant CYP119 enzyme was cloned by site-directed mutagenesis. L69G and WT CYP119 was expressed successfully in Escherichia coli BL21 (DE3) cells with isopropyl β-D-1- thiogalactopyranoside (IPTG). This study shows that L69G mutation is important for binding to progesterone. This was predicted by in silico mutagenesis in a previous computational study. Isolation and purification of the WT and L69G CYP119 were carried out. Activity assays and substrate binding studies of the enzymes were performed and compared each other. L69G mutation did not cause significant effect on Amplex Red® oxidation and styrene epoxidation activities. L69G CYP119 (KS: 34.55 ± 7.4 μM) showed higher affinity for progesterone compared to WT CYP119 (KS: 69.8 ± 48.9 mM). A new product, thought to be hydroxylated progesterone, was formed as result of hydroxylation of progesterone by L69G CYP119 using peroxidase shunt pathway.Master Thesis The Rational Design of a Novel Biocatalyst Using the Heme-Nitric Oxide/Oxygen Binding Protein(Izmir Institute of Technology, 2017) Meşe Özçivici, Gülistan; Meşe Özçivici, Gülistan; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyRecent advances in recombinant DNA technology and protein design have led to the application of biocatalysis as an alternative to chemical catalysis in the synthesis of enantiopure products due to high regio- and enantioselectivity. Hemeproteins are proteins with a heme prosthetic group that play diverse roles in biological systems, making them good candidates for biocatalysis. The Heme-nitric oxide/oxygen binding (H-NOX) protein was identified by homology to the soluble guanylate cyclases. Here, the H-NOX domain from the methyl-accepting chemotaxis protein, Thermoanaerobacter tencogenesis (TtH-NOX), was tuned into a biocatalyst using rational design. Four variants of TtH-NOX were cloned, purified and characterized. Each variant was then tested for their catalase and peroxidase activities. The wild type TtH-NOX inefficiently catalyzed the hydrogen peroxide decomposition (catalase activity) and 2,2’-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid (ABTS) oxidation (peroxidase activity). However, the Y140H mutant exhibited an efficient five-fold increase in catalase and peroxidase activities as compared to the wild type. The other mutants, H102Y, H102C and Y140A TtH-NOX, were not good catalysts for both reactions. Therefore, the mutations resulted in changes in reaction rates and electronic properties of the heme group. The mutations affected the molecular mechanism of the hemeprotein, showing that both the proximal and distal pocket residues are vital for catalysis. However, the mutation of the distal tyrosine to histidine of TtH-NOX has significantly improved its catalytic activities. These observations contribute to the understanding of the physiological roles of hemeproteins. This project could also lead to discovery of novel biocatalysts and aid in the design of future biocatalysts.